Centrifugal fast chromatograph

ABSTRACT

A centrifugal fast chromatograph is disclosed. The apparatus includes a rotor, a rotor drive, a plurality of chromatographic columns mounted on the rotor, a transfer disk mounted on the rotor, photodetecting apparatus, a gradient maker and a microprocessor control system. The microprocessor controls and monitors the entire chromatograpy process. The centrifugal fast chromatograph permits the performance of multiple, rapid, and simultaneous separations.

This application is a continuation of application Ser. No. 210,160,filed 6/9/88 now abandoned which is a continuation of Ser. No. 065,590,filed 6/23/87 now abandoned.

TECHNICAL FIELD

The present invention relates to an improved centrifugal chromatograph.In particular, the present invention relates to a centrifugalchromatograph wherein multiple separations may be performedsimultaneously, rapidly, and relatively inexpensively.

BACKGROUND OF THE INVENTION

High-pressure, high-resolution liquid chromatography has come into wideuse to separate proteins, nucleic acids, metabolites, drugs, and a widevariety of compounds both in research laboratory, in industry, and inclinical laboratories. The systems now in use are expensive, and includenon-pulsating high-pressure pumps, valving (often operating at highpressure) for sample introduction, high-pressure prepacked columns,small uniform beads adapted to achieve high resolution separations, aspectrophotometer or colorimeter to monitor each column, and a fractioncollector for each column as essential components. In addition,microprocessor systems for analysis of the chromatographs, programs tointegrate peaks, and a printer to print out quantitative results arealso used. The systems do not include positive means to preventanomalous flow or channelling to insure ideal flow through the column,to positively remove air bubbles, to prevent a column from running dryduring use, to prevent anomalous flow and mixing either in the headspaceabove the column where flow fans out from the narrow-bore inlet tubingto the full bore of the separations column, or where the converserestriction in diameter occurs as liquid leaves the column and isconstricted into the detector flow cell. In addition, the rapid dataprocessing capabilities of microprocessors are not fully used; althoughone microprocessor can now process, manage, and display data from adozen or more columns, microprocessors are not so used.

In presently available systems the entire collection of componentsservice, drive, and monitor only one column at a time, can perform onlyone analysis at a time, is expensive, is subject to a variety ofoperational problems which may produce anomalous results, and producesresolution lower than that which should, theoretically, be obtained.Additionally, packing new columns or repacking with cleaned columnseparation materials is difficult if not impossible in the field.

One recent development has altered the instrumental requirements forhigh resolution separations. This is the development of very uniformspherical beads or resins which reduce the back pressure required toachieve high resolution. Where thousands of pounds per square inch ofpressure have been required previously, only hundreds are required now.This means that the pressure requirements can be met in a centrifugalfield at much lower speeds. The mechanical strength of the beads hasalso increased to prevent their deformation under pressure. This higherstrength also produces resistance to deformation in centrifugal fields.

Prior noncentrifugal chromatographs encountered numerous other problems.There was no automatic compensation for flow resistance in differentcolumns. When a number of parallel columns are fed from one pump in anon-centrifugal system, the flow through different columns will beslightly different depending on differences in flow resistance inindividual columns.

In column chromatography there has not previously been a method forpreventing anomalous flow such as channeling through the packing. If thepacking is uneven, or if the packing particles are of different sizes,the resistance to flow in different parts of the column will bedifferent. Liquid will flow through the lines or channels of leastresistance thereby creating uneven flow. The flow distortions andchanneling decrease resolution. Where there are many small anomalies,band widening is observed. Where the anomalies are large, band tiltingoccurs. Sharp bands which are tilted are also observed as broadenedpeaks during elution. Extraordinary efforts have gone into theproduction of column packings of spherical particles of uniform diameterto minimize microanomalous flow. The vastly improved resolution obtainedwith homogeneous, uniform, particle-sized beads demonstrates the keyimportance of micro-flow control and minimization of channeling.However, homogeneous particles do not positively prevent anomalous flow.Even with homogeneous particles, the packing may be uneven with localparticle compression, or clogging due to the formation of precipitatesin the packing may occur. Both conditions produce anomalous flow.

Resolution is also lost during flow through tubing into the column(laminar mixing), during radial flow expansion as the fluid flows fromthe small bore line leading into the column to the wide bore column,during the decrease in cross-sectional area at the bottom of the column,and by laminar mixing during flow to and in the optical flow cell andthe intervening tubing.

Arranging a constant column path length was also difficult. The fluidflowing through the chromatographic system may be considered as beingcomposed of many small fluid elements. In free fluid flow these may bekept in order by centrifugal force acting on a density gradient.However, the solutes in these fluid elements are retarded to differentdegrees by flow through the separative column packings, and if the pathlength through the packing is different for different solute elements,considerable loss of resolution occurs. If there is a free space at theends of the columns, considerable mixing occurs in the absence of agradient combined with a centrifugal field.

If there is no free space at the ends of the columns and the columnsmerely constrict to a small diameter at each end, then fluid flowingalong the edge of the column will flow through a longer path than doesfluid flowing along the center axis of the column, resulting in loss ofresolution.

Finally, in conventional chromatography careful attention must be paidto eliminating air bubbles and preventing their formation. In someinstances degassing of eluting solutions is required.

These were just some of the problems encountered by priorchromatographs. These, and other problems have been addressed by thecentrifugal fast chromatograph of the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide means for performingion exchange, gel filtration, affinity chromatography, andchromatographic separations of any sort rapidly, in multiple parallelarrangement, efficiently, and relatively inexpensively.

The above and other objects are achieved by the centrifugal fastchromatograph (CFC) of the present invention. The CFC automaticallyperforms, monitors, and compares multiple sample elutions, andregenerates the chromatograph columns. The invention also includes amethod of simultaneously conducting gradient elution of a plurality ofsamples. The CFC includes a chromatograph rotor, and a rotor drive. Aplurality of chromatographic columns are equiangularly mounted on thechromatograph rotor and each chromatographic column has an optical flowcell with transparent windows. A sample holding transfer disk is locatedat the center of the chromatographic rotor. The transfer disk hasplurality of sample wells equiangularly disposed around its periphery.Each sample well corresponds to a respective chromatographic column.Stream apportioning blades are equiangularly disposed on the inner edgeof distributor rings of the chromatograph rotor and direct solution fromsample wells into the chromatographic columns. The stream apportioningblades also apportion the eluting gradient into the chromatographiccolumns. Two fluid carrying lines extend from the region of the transferdisk to each chromatographic column. Both have valves to direct fluid toan exit drain. One line is connected to the inner end of thechromatographic column and the other line is connected to the outer end.A light source is mounted on one side of the chromatograph rotor nearthe periphery and is oriented to shine a light beam through the opticalflow cells. A photodetector is mounted above or below the chromatographrotor opposite the light source to detect light shone through theoptical flow cells. A gradient maker is in fluid communication with thechromatographic columns through a distributor ring containing streamapportioning blades which distributes eluting gradient into allchromatographic columns simultaneously through individual column feedlines. A microprocessor type computer coordinates the operation of theCFC. The microprocessor controls the gradient maker and the rotor drive.It controls the chromatographic process, monitors the chromatographicprocess and provides real time CRT displays of all chromatographiccolumns. It also controls an automatic pipetter used to load samplesinto the transfer disk.

The CFC of the present invention includes a microprocessor which bothcontrols and monitors the separations. A cathode ray tube (CRT) displaysthe chromatographic separations in real time, while the gradient makerproduces the gradient from solutions and controls column regeneration.At the start of an analysis the samples are loaded on the transfer diskin a disk loader. The transfer disk is loaded into the chromatographrotor at the start of a run, and the rotor is accelerated by the rotordrive under control of the microprocessor. The samples are transferredby centrifugal force from the transfer disk, between respective matchingstream apportioning blades, and into their respective column feed lines.Since the transfer disk and the apportioning blades attached to thedistributor ring all rotate together, each sample flows uniquely betweenone matching pair of blades and into one column. The eluting gradientthen flows against the same blades, and is apportioned to follow thesamples into the column feed lines. Effluent from the rotor may beeither led to a drain or collected in a corotating fraction collectionring. Absorbance of the effluent stream is measured by the light sourceand the photodetector which provides the signal to the microprocessor.The rotor position during rotation is determined using a synchronizationsignal pickup. Two distributor rings with apportioning blades, onedirectly above the other, are used. One distributes the eluting gradientduring analysis, and the other distributes the regeneration andequilibration solution. An inwardly-projecting lip between the two ringsprevents crossflow between the rings.

Operation of the centrifugal fast chromatograph of the present inventionprovides the required hydrostatic pressure for column packing,chromatographic separation, column regeneration, and sample loadingusing centrifugal force. Rotation apportions one liquid stream intomultiple columns and also provides chopping of the single sensing beamor beams required to achieve reproducible absorbance measurements. Thecentrifugal fast chromatograph has traps to prevent any column fromrunning dry at rest or during rotation, and valving to control thedirection of flow through the columns during analysis and columnregeneration. Two types of traps are integral to the design. The firsttype prevents the columns from draining dry at rest. It includesupwardly-sloping center and edge lines which connect the inner and outerends of the columns through valves to the central distributor rings. Thesecond type functions during rotation and includes the column and theedge line connecting the column inward to the valve. If liquid feed isstopped during rotation, liquid will drain only up to the open valve andthe column will not run dry. If flow is restarted during rotation, airin the feed line will be displaced rapidly by centrifugally forcedliquid; the air will move centripitally.

Separated fractions may be collected at intervals and the hydrostaticpressure on the columns may be changed by either changing the rotationalspeed or the length of the fluid column inward of the separation column.Density gradient elution in the centrifugal field positively stabilizesflow through the system and keeps sample and separated zones fromtilting or being widened by anomalous flow. A microprocessor including aliquid gradient generator used with the centrifugal fast chromatographcontrols the entire analytical procedure. The centrifugal fastchromatograph may include means for introducing the entire elutiongradient into the rotor at one time by enlarging the chambers in thedistributor ring, and then for allowing it to flow through theseparations columns without additional fluid introduction. Additionaloptional means for introducing samples and elution gradients into eachcolumn unit separately and individually during rotation may be included.Further optional means for driving the rotor using a printed motorincorporated into the rotor itself may be used. A printed motor has itselectrical lines and windings "printed" onto plastic-coated metal whichmay be integral with the rotor. Printed motors can withstand the forcescreated in stronger centrifugal fields. The direction of liquid flowduring elution may be either centrifugal (referred to as System A) orcentripetal (referred to as System B). The preferred direction iscentrifugal.

Thus, the centrifugal fast chromatograph, in a single apparatus,includes means for moving premeasured samples from a transfer disk intoeach of several columns aligned with the sample chambers of the transferdisk, means for apportioning one unpressurized gradient stream equallyinto all columns, and means for providing the hydrostatic pressurerequired to achieve the separations using centrifugal force. Means formeasuring the light absorption of the effluent stream from all columnsduring rotation and means for collecting fractions from all columns (orfor rejecting the effluent stream) are also provided. The presentinvention also includes means for regenerating all of the columns and,if necessary, monitoring regeneration. Means for positively keeping allzones and boundaries essentially perpendicular to the direction ofradial flow and for positively preventing channelling and anomalous flowduring gradient elution, means for preventing the columns from runningdry either during rotation or at rest, and means for controlling flow sothat all parts of a sample or separated zone travel through exactly thesame distance in the separation medium during flow between sampleapplication and detection are also included. The centrifugal fastchromatograph also has means for insuring that no air bubbles areretained in the analytical stream, means for collecting samples atpredetermined intervals or for rejecting to waste portions of the streamwhich are of no interest, and means for packing or repacking columns inposition.

Flow during elution may be either centrifugal (radially outward), orcentripetal (radially inward), with flow in the opposite directionsduring regeneration. Most of the design descriptions discussed belowconcern a rotor system (System A) with outboard elution flow, and centerflowing column regeneration.

The types of analysis which may be performed on the centrifugal fastchromatograph include but are not limited to the analysis of humanplasma proteins, nucleotides and their derivatives, amino acids andtheir derivatives, urinary metabolites, therapeutic drug monitoring,monitoring for drugs of abuse, and the analysis of the proteins of wheatand other seeds.

Various additional advantages and features of novelty which characterizethe invention are further pointed out in the claims that follow.However, for a better understanding of the invention and its advantages,reference should be made to the accompanying drawings and descriptivematter which illustrate and describe preferred embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a centrifugally driven gradient elutionchromatographic system according to the present invention.

FIG. 2 is a side view of a centrifugally driven gradient elutionchromatographic system according to the present invention.

FIG. 3 is a top view schematic diagram of the chromatograph rotor of thecentrifugally driven gradient elution chromatographic system.

FIGS. 4 A-J illustrate the operational steps of the chromatographyrotor.

FIG. 5 illustrates inward elution flow in the chromatography rotor.

FIGS. 6 A-D illustrate how gradients are used to control flow throughlong path flow cells.

FIGS. 7 A-D illustrate how the path length is controlled in thechromatographic column through column packing.

FIGS. 8 A-D illustrate a piston operated valve system used with thecentrifugally driven gradient elution chromatographic system of thepresent invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The central fast chromatograph of the present invention is acentrifugally driven gradient elution chromatographic system. Thissystem is shown diagramatically in FIG. 1. A microprocessor 10 bothcontrols and monitors the separations. Cathode ray tube (CRT) 12displays the chromatographic separations in real time, while gradientmaker 14 produces the gradient from solutions 16 and 18 and controlscolumn regeneration. At the start of an analysis the samples are loadedon a transfer disk 20 (shown in FIG. 2) in disk loader 21 which is anautomatic pipetter known in the art. Transfer disk 20 is loaded intochromatograph rotor 22 at the start of a run, and the rotor isaccelerated by drive 24 under control of microprocessor 10. Effluentfrom the rotor 22 may be either led to drain 26 or collected incorotating fraction collection ring 28. Absorbance of light by theeffluent stream from light source 30 is measured using photodetector 32which provides the signal to microprocessor 10. The rotor positionduring rotation is determined using synchronization signal pickup 34.

FIG. 2 illustrates a side view of the system. A control center includescontrol panel 35, microprocessor 10, CRT display panel 12, keyboard 37,speed and temperature indicators 39, and gradient maker 14. Centrifugalchromatograph rotor 22 is driven by rotor drive 24 which is controlledby the control panel of the control center. Chromatographic columns 36are mounted for rotation on centrifugal chromatograph rotor 22. Thesample-holding transfer disc 20 is mounted in the center of rotor 22.Optical flow cells 38 having transparent windows 39 are mounted oncentrifugal chromatograph rotor 22. Light source 30 is mounted oppositephotodetector 32, which may be photomultiplier 40, to measure absorbanceof the effluent stream. Upper or edge line distributor ring 42 for edgeline introduction of the regenerating solution is disposed at the end ofsolution feed line 44, which runs from gradient maker 14 on the controlcenter 35. Edge lines 46 are fluid carrying feed lines having anantiregurgitation slope disposed between upper distributor rings 42 andedge line values 48. Lower or center line distributor ring 50 for centerline introduction of the regenerating solution is disposed at the end ofsolution feed line 62, which runs from gradient maker 14 on the controlpanel 35. Center lines 52 are fluid carrying feed lines having anantiregurgitation slope and are mounted between the center end ofchromatographic columns 36 and distributor rings 50. A center line valve54 (FIG. 3) is disposed on each center line 52 at the center end ofchromatographic columns 36. An edge line valve 48 is mounted on one endof edge line drain 56. Drain and fraction collecting ring 28 is mountedon the other end of edge line drains 56 and includes collecting tubesand collecting ports 58 leading to stationary drain collecting ring 60.Stationary drain collecting ring 60 is mounted on the outside of rotor22 to collect fluid drained from the rotor 22. Feed line 62 runs fromgradient maker 14 to center line distributor ring 50 which is used toelute chromatographic column 36.

A horizontal section looking down through the System A centrifugallydriven gradient elution chromatograph rotor is shown in FIG. 3.Chromatograph rotor 22 includes a sample holding transfer disk 20 inwhich a plurality of sample wells 64 are placed. Sample wells 64 areplaced around the outer edge of transfer disk 20 and correspond torespective chromatographic columns 36. Chromatographic columns 36 areconnected to the periphery of transfer disk 20 by center lines 52.Stream apportioning blades 66 in center line distributor ring 50 guidethe fluid sample between transfer disk 20 and center lines 52. Centerline valves 54 are disposed on center lines 52 to divert material towaste during reverse flow during column regeneration. Optical flow cells38 having transparent windows 39 are disposed on the outer end ofchromatographic columns 36 adjacent the connection to edge lines 46.Edge line valves 48 are disposed on edge lines 46 to divert material towaste during elution. Two coaxial stream segmentation devices ordistributor rings, upper distributor ring 42 (not shown in FIG. 3) whichfeeds the edge lines 46 and lower distributor ring 50 which feeds thecenter lines 52, are used to apportion the stream into multiple columns

The chromatographic system of the present invention has superior flowcharacteristics which result in higher resolution. Outward flow duringelution (System A), which is preferred, requires that the sample beplaced at the center end of the chromatographic columns 36, that theoptical flow cells 38 or other detector be at the outboard end of thecolumns 36, and that elution be done with a gradient of decreasingphysical density. It also requires that the liquid in the column 36 atthe start be denser than the sample or the starting (heavy) end of theelution gradient. The implications of these special requirements arediscussed below.

When center elution flow is used (System B), the fluid in the column atthe start is less dense than either the sample or the starting end ofthe elution solution introduced through the edge line. The elution fluidexits through center line valves 54 on center lines 52, and the elutiongradient increases in physical density from start to finish. The sampleis applied to the bottom of the columns 36, through the edge lines 46,exits through center line valves 54 on center lines 50, and the opticalflow cells are at the top (center end) of the column 36 as shown in FIG.5. It is feasible to construct a system that will work either way, andhas flow cells at both the tops and bottoms of the columns 36.

The sequence of events during columns packing, column regenerationand/or equilibration, and analysis are shown diagramatically in FIG. 4,where the same column is presented in a series of steps involving columnpacking, regeneration, and analysis. The valves 48 and 54 may be set toallow change in direction of flow. Specifically, during column packing,the suspension being packed flows through the lower stream-segmentationdevice (distributor ring 50) and into the center lines 52, past theclosed center line valves 54, and into the columns 36. Liquid flowingout of the columns 36 past the lower frit flows through the optical flowcells 38, and to the open edge line valves 48 to drain. When columnpacking is complete, a small plug may be introduced into the centerlines 52, and moved into position by centrifugal force. This preventspacking material from moving and resettling when the rotor 22 is atrest. Sloping of the center and edge, 52 and 46, respectively, lines isused to prevent fluid from flowing out of the columns 36, flow cells 38,and outer loop when the rotor 22 is at rest.

In FIG. 4A a suspension of inert particles 70 is introduced intochromatographic column 36 through center line 52. Both edge line valve48 and center line valve 54 are set for outward flow, i.e., valve 48 isopen and valve 54 is closed. The inert particles 70 pack the lower coneof chromatographic column 36. The active packing 80 which produces thechromatographic separation is then introduced and, in FIG. 4B,chromatographic column 36 has been partially packed due to centrifugalforce. In FIG. 4C, all of the active packing 80 is in place, and theupper cone of chromatographic column 36 has been partially packed withinert material 70. In FIG. 4D, chromatographic column 36 has been fullypacked and an upper porous plug 72 has been set in place in center line52 above the chromatographic column 36. In FIG. 4E, edge line valve 48and center line valve 54 have been set for inward flow i.e., valve 48 isclosed and valve 54 is open. During rotation, a washing or regenerationor equilibration liquid 74 is introduced into edge line 46 through upperdistributor ring 42 thereby causing column regeneration orequilibration. Liquid density is controlled so that the a liquid whosedensity is greater than that of the dense end of the elution gradient isleft in place in the column 36. A sample 76 is placed in transfer disk20 adjacent the center line 52 in FIG. 4F while the rotor 22 isstationary. The sample is less dense than the liquid in chromatographiccolumn 36. The edge line valve 48 and center line valve 54 are, onceagain, set for outward flow i.e., valve 48 is open and valve 54 isclosed through chromatographic column 36. In FIG. 4G, the rotor 22 isaccelerated and the sample 75 is transferred centrifugally into therotor 22 and then into center line 52. During continued rotation, aneluting gradient 77 is introduced to the rotor center line 52 as shownin FIG. 4H. Gradient elution of chromatographic column 36 has begunalthough no separation of the sample 76 occurs in the inert particles 70in the upper cone of chromatographic column 36. In FIG. 4I, constituents1, 2, 3, and 4 of the sample have separated in chromatographic column36. In FIG. 4J, peaks or bands 1, 2, 3, and 4 representing eachrespective constituent of the sample are eluted sequentially through thecuvet 79 outboard of chromatographic column 36. The peaks move throughthe cuvet 79 without backmixing. On completion of the analysisillustrated in FIG. 4, column regeneration by back flow occurs and asolution having a density greater than the dense end of the elutiongradient is left in place. The analysis may be repeated by beginningwith the step illustrated in FIG. 4F.

When the flow is backward through the column 36 during columnregeneration and equilibration (hereinafter referred to as System A),liquid flows in through the upper stream-segmentation device(distribution ring 42), out through the edge line 46, past the closededge line valves 48, to the centrifugal (outer) end of the column 36,and inwardly through the optical flow cells 38, and column 36, and outthrough the open inner line valves 54 to drain (or collection) 60.

The location of the optical flow cells 38 when the elution flow is in aninward or centripetal radial direction during analysis (hereinafterreferred to as System B) is shown in FIG. 5, with the optical flow cells38 located centripetal to (above) the column 36 on center lines 52. Insystem B, the eluting solution flows through edge line 46 tochromatographic column 36. The pressure head during elution isproportional to the distance D to center line valve 54.

System B has an inherent problem with density reversal in the edge lines46. At the start of an analysis the least-dense fluid used fills thesystem. The sample is adjusted to be slightly denser, and will mixrapidly with fluid in the edge lines 46 as it is introduced. Once thesample has passed the hairpin loop at the edge, and begins inward flow,density-gradient stabilization begins to be effective. As the gradientflows into the rotor 22 during analysis, constant backmixing occurs inthe edge lines 46, followed by stable flow in the columns 36, flow cells38, and center lines 52. A solution to the problem of backmixing duringsample introduction is to inject the samples individually through aself-sealing rubber port 81 at the centrifugal end of the columns 36with the rotor 22 at rest. This solves the problem, but makes the systemless desirable from the automation viewpoint.

During sample loading followed by introduction of the gradient, flow andvalve settings are as shown in FIG. 5. Column regeneration involves flowof solution in through center line 52, and drainage out through the exitport of valve 48, i.e., the flow and valve settings are the same as forcolumn packing.

The CFC has automatic compensation for flow resistance in differentcolumns that maintains constant flow. In the CFC, the actual pressurehead on each column is a function of the length of the liquid columncenter of the exit line. If one column has slightly less resistance toflow than the other columns, fluid will flow more rapidly, the length ofthe liquid column will decrease, there will be less driving pressure,and flow through that column will automatically adjust itself to matchthe others, provided that the liquid gradient is not introduced toorapidly. The upper portion of the column center of the exit line can beexpanded to include a large volume of fluid, up to and including theentire gradient. In a centrifugal field large density gradient arestable. Air bubbles will not impede flow as air bubbles are forcedinwardly by the centrifugal field.

During elution with either System A and System B, the gradient used is abifunctional gradient, varying first in density or specific gravity tocontrol flow and maintain fluid segments in their proper sequence, andsecond in eluting power, to sequentially elute components of the mixturebeing analyzed from the column.

With System A, the regeneration or equilibrium liquid 74 in the rotor,the sample 76 and the elution gradient 77 must be arranged to be inorder of decreasing density. For much protein and other work involvingreverse phase chromatography, the gradient 77 is one of increasingacetonitrile concentration. Acetonitrile has a density or specificgravity of 0.785 g/ml; hence a gradient from water or a customary buffersolution will grade from a density of approximately 1,000 g/ml to 0.785g/ml. The specific gravity will decrease as required for System A. Ifsucrose or other inert gradient material is added to the equilibrationsolution 74, optionally to the sample 76, and to the denser of thesolutions used to make the gradient 77, a steeper density gradient maybe obtained.

When the gradient 77 normally used is one of increasing saltconcentration and is therefore of increasing density, an inertdensity-increasing material must be added to the equilibration solution,in lesser amount to the sample 76, and must be in decreasingconcentration in the gradient 77 to yield a gradient of decreasingphysical density, but of increasing elution power. This is onedisadvantage of System A when used with columns and elution proceduresrequiring very concentrated salt solutions. With System B, whichrequires a gradient 77 of increasing density, gradients which arenatively of increasing density may be used directly.

The disadvantage of System B is sample-zone smearing during flow throughthe edge lines 46. This may be minimized by having very small bore edgelines 46. The smearing may be of little effect in instances where thesample 76 is strongly absorbed in a small volume of column packingmaterial 80 at the start of flow through the columns 36, therebyreconcentrating the sample 76 at the outset into a small volume.Injection of the sample 76 at the rotor edge initially also solves thisproblem but introduces operational disadvantages.

An advantage to System B is that flow is counter to centrifugal force.Therefore flow is tending to resuspend the column packing, whilecentrifugal force is tending to pack it down. This sets a limit to theflow rate without positive positioning of an upper frit. With System A,flow and centrifugal force are in the same direction, hence flow andcentrifugal force will combine to pack down the column material 80 more.This is a disadvantage with column packings 80 which are deformable, andcan be overcompressed causing flow reduction.

With the systems described here, which use density gradients,extraordinary control of flow is achieved by centrifugal force. This isillustrated by considering the effect of 0.001 increment in density in afield of 5,000 g. This produces an effect equivalent to a difference indensity of 5 grams/ml.

The stabilizing effect of the gradient will also be seen in the opticalflow cells 38. To increase the sensitivity of detection, a long opticalpath is desired. This is often attained by using a cylindricalmicro-bore flow cell as shown in FIG. 6A in which liquid fromchromatographic column 36 flows into optical micro flow cell 38. Theliquid is illuminated by light from light source 30 which passes throughwindows 39. In static systems, this often presents cleaning problems,and becomes useless with the presence of small air bubbles. In addition,resolution is lost by laminar mixing. These problems are not a factorwith the centrifugal fast chromatograph. If a long-path flow cell 38with a reasonable optical cross-section is arranged as shown in FIG. 6B,then, when no gradient is present, laminar mixing will greatly increasepeak width and decrease resolution. However, with centrifugal gradientstabilization, no mixing occurs as shown in FIG. 6C. The zone or peak100 is stabilized as it passes through the flow cell 38. The peak 100flows through the flow cell 38 without an appreciable volume change, andthe resolution obtained in the column 36 is not degraded duringdetection. FIG. 6D is an end view of the flow cell 38 of FIGS 6B and 6C,illustrating the very narrow one dimensional cross section.

The CFC provides constant column path length. This overcomes resolutionloss as illustrated in FIG. 7A-D. FIG. 7A-D shows how the path length iscontrolled as the eluting gradient 77 moves through the column packing80, and how mixing in a free space at the ends of the column 36 isovercome by the centrifugal field. In FIG. 7A, a fully packed columneluted at rest is shown. Different fluid elements traverse differentdistances. Peaks from paths 1 and 2 are graphically shown along withresolution loss as all paths are summed. In FIG 7B, the column is alsoeluted at rest. Mixing occurs in the free space above the upper frit andthe free space below frit 78. Thus, as shown, the separation distancefor all paths is identical, but resolution is lost in the free spaces atthe ends.

If the cones at the end of the column 36 are filled with inert material70 which does not contribute to the separation, then in a centrifugalfield the gradient 77 and centrifugal force will stabilize zones withinthe cones so that they widen evenly in the cones, and little resolutionis lost. All fluid elements having the same density then pass throughidentical distances in separative packing 80 as shown in FIG. 7C.Retarded solute zones, shown as dark bands, are therefore kept sharp anddo not tilt.

If strong frits are available which will withstand the centrifugalforce, then an open-cone construction can be used at the bottom of thecolumn as shown in FIG. 7D. The frit shown at the top may be a plug heldin place by centrifugal force. Flow is controlled by the gradient in thecentrifugal force field in the cones and in the body of the column, andseparated zones are kept perpendicular to the direction of centrifugalforce as shown, and high resolution is achieved.

With the CFC there are no "wall effects" such as occur when particlesare sedimented in a centrifugal field. Therefore, the separation chamber(i.e., the column 36) can have almost any configuration including thatof a cube, a cylinder, a sector, or an inverted sector.

In the CFC, gradient overloading which may cause mixing and inversionswill rarely occur except at the original sample zone or early in thecourse of elution. Hence the greatest density slope must occurimmediately under the sample 76 initially, and later in those parts ofthe elution gradient 77 containing the most sample mass. The "turnovereffect" which is well known in density gradient centrifugation and isdue to differential diffusion of the sample solution 76 down into thegradient 77, and the diffusion of the gradient solution 77 back up intothe sample 76 will not be a problem in the chromatograph rotor becauseof the short time between sample introduction and initiation ofseparation which minimizes diffusion, and because the differences inmolecular mass between the gradient 77 solution and the sample 76 arenot as large as in the zonal centrifuge separation of subcellularparticulates.

Air bubbles are not a problem in centrifugal chromatography where thecentrifugal fields are sufficiently large to move any air bubblescompletely out of the column 36 and flow lines 46 and 52. Thus, even invery small bore lines filled with air, samples will flow past the airand quickly displace it centripetally. Also, because gas solubility is afunction of pressure, should any air bubbles (or other gas bubbles)occur, they will tend to redissolve rapidly given the pressures existingin the rotor during rotation.

The design of the system prevents the columns 36 from ever running dry.The exit lines 46 and 52 are all center of the columns, and are tiltedup at some point along their length. Providing the exit lines 46 and 52inboard of the columns 36 prevents their running dry during rotation.Traps are thus provided in the lines as an integral part of theirdesign.

The gradient maker 14 may be of a positive type using differentiallydriven pumps to mix two or more liquids to produce a liquid gradient, ora passive type in which two containers having complementary geometriesare drained by gravity through a mixer to form a gradient. These arewell known. One or more liquids are also required for columnregeneration, and these are introduced by reverse flow through thecolumns after adjusting either manually or automatically the feed lineand drain line valves 48 and 54. The gradient 77 or the regenerationsolutions 74 flow into the centrifugal chromatograph during rotation.

The methods for signal acquisition, data processing, signal averaging,data display, and the analysis of chromatographic data are well known.

In the designs described, the driving forces are generated by thedifference in radius of the center (centripetal) fluid column (r1), andthe radius of the drain exit (r2), as shown in FIG. 5. Centrifugal forceis directly proportional to radius. Hence, the pressure generated bythis difference in radius is equal to the average r=(r1+r2)/2 multipliedby the centrifugal force, multiplied by the liquid density, andmultiplied by the column length. If the pressurizing column extends from4 cm to 20 cm (r1 and r2), the centrifugal force is calculated at 12 cm,and the pressure column is 16 cm long. At 3000 rpm the centrifugal forceat 12 cm results in a pressure head of 19,440 g/cm², or 276.5 psi. At4000 rpm the centrifugal force at 12 cm results in a pressure at thebottom of the pressure column of 33,400 gm/cm², or 489.28 psi.

A variety of methods for operating the two valves attached to eachcolumn are possible. Several are discussed below. In the illustrationsprovided thus far, the two valves are operated separately and manually.It is evident that since the valves are always operated simultaneouslythat each pair may be interconnected, and that all valves may beoperated by one mechanism. The valves may be pressure operated,centrifugally operated, electrically operated, or mechanically operated.

Pressure operated valves are illustrated in FIGS 8A-C. When one of thelines 46 or 52 attached to the column 36 is full of liquid, the other 46or 52 is empty out to the exit valve 48 or 54. Hence there isconsiderable hydrostatic pressure in one line. If liquid suddenly runsthrough one set of lines during rotation, pressure sufficient to closethe valve attached to that line is created. By continuing flow into theline with the closed valve, the valve is kept closed. When liquid flowin ceases, the liquid level will gradually drop to equal that of theother line (i.e., the exit valve level), and sudden liquid introductioninto the opposite line will reverse the valve setting. Operating detailsare shown in FIG. 8 using a piston operated valve. FIG. 8A is a sidesectional view of the chromatograph rotor 22 showing the position ofpiston operated edge line valves 48. In FIG. 8B, a piston operated valve48 or 54 (used for both center lines 52 and edge lines 46, is its restposition preparatory to column elution. Piston 84 has two head portions85 and 87 and spring 86 biasing piston to the left. Piston 84 has twoopenings 88 and a 90 through its center to permit passage of liquid.First opening 88 provides a fluid passageway from center line 52 todrain 92 and second opening 90 provides a fluid passageway from edgeline 46 to drain 94. When piston 84 is driven to the left, the edgeline-drain connection 90 is open and the center line-drain connection 88is closed. In FIG. 8C valve is in position for column regeneration. Edgeline 46 is filled during rotation and exerts a pressure on the left ofpiston 84 sufficient to drive piston 84 to the right, thereby closingedge line 46 to drain 94 and opening center line 54 to drain 90. FIG. 8Dis an end view of piston 84 showing the opening 88 in the center.

In centrifugally operated valves, centrifugal force either alone or incombination with hydraulic pressure is used to operate the valves.Spring loaded valves are well known, and can be arranged so that theyare in one position at rest (due to spring loading), and in thealternate position in a centrifugal field where centrifugal forceopposes the spring. Spring tension can be set so that position changeoccurs at relatively high speed, making it possible to regeneratecolumns at one speed, and elute at a second. It is also feasible tocombine centrifugal operation with hydrostatic pressure-operated valvesso that once the positional change occurs, hydrostatic force maintainsthat position.

In electrically operated valves an electric drive system is used in thechromatograph rotor to change all valve positions at once.

In mechanically operated valves, the valves are arranged so that theyare operated by mechanical movement. For example, the valves may bearranged so that the members of a pair are one above the other, onshafts that extend above and below the rotor. With a vertically-movingnon-rotating ring all of the extensions may be simultaneously pusheddown from above, or with a similar ring placed below the rotor, all maybe pushed up.

Detection of valve operation failure may be important in routine longterm operation. If the edge and center line drains open into separatedrain collection rings (not shown), then it is a simple matter todetermine that fluid flow exists from one ring, and is absent from theother. If flow occurs from both collector rings, then valve failure hasoccurred.

The center line can be enlarged, especially at its inboard end, to holda considerable volume of liquid, in the limiting case, the entireelution gradient volume.

Because the system will not run dry during rotation, there is no lowerlimit for the rate of feed of elution buffer. However, if the elutionfeed rate is to high, liquid will flow out of the center linedistributor ring. In one modification of the system, this liquid isguided to the center line collecting ring and is detected. The elutionflow rate may then be increased until fluid is detected in the centerline collecting ring, and then decreased until the rate of elutionbuffer outflow matches inflow. Similarly, the edge line distributoroverflow may be guided to the edge line collecting ring which normallycollects no fluid during regeneration, and excess flow duringregeneration detected.

For most applications fractions are not collected, and the fractioncollector described here is omitted, and the exit lines are led directlyto the drain collecting ring. However, when fraction collection isdesired, then a fraction collector ring 84 is provided which corotateswith the chromatograph rotor of the CFC but can be indexed around tocollect separate fractions from each chromatographic column 36. Theinitial setting of the fraction collector ring 28 is designed so thateach exit line drains through the collector ring 28 to the drain ring 60and to waste. This allows column regeneration, and also allows theinitial part of the elution gradient to be discarded.

The fraction collector ring 28 may be designed to receive tubes 58, butit is more optimally designed to hold flattened collection chamberswhich allow a very much larger number of collection units to becompressed into the limited space of the collector ring 28. Tubes orflattened collection chambers are angled so that they do not spilleither at rest or during rotation.

The position of the rotor 22 during rotation is sensed by a smallelectromagnetic or optical pickup positioned next to the chromatographrotor 22, and a similar pickup determines the position of the collectorring 28. Using a suitable algorithm the position of the ring 28 relativeto the exit lines is determined, and the identity of the collectionvessels being used is known.

To advance the collecting ring 28 one unit (one tube or collectingvessel), a variety of mechanical, electrical, or hydraulic mechanismsmay be used. The preferred method uses a mechanical brake to apply asmall force to the collecting ring 28, retarding it relative to thechromatograph rotor 22. A ratcheted escapement mechanism allows thecollector to move only the width of one collecting vessel. The rachet isthen operated using either downward force applied through a ring abovethe rotor, electrical force, or a retarding force applying pressure to aspring which resets the ratchet when the retarding force is released.Escapement mechanisms suitable for this purpose are well known.

It is feasible to use a large seal of teflon or other self lubricatingmaterial attached to a central bell-shaped chamber to isolate the centerof the rotor from the rest of the rotor chamber, and to keep the centerat atmospheric pressure while a vacuum is produced in the rotor chamber.This produces an additional driving pressure of one atmosphere acrossthe column in the rotor, and also allows the contents of the collectingvessels to be concentrated by evaporation of lyophilization in acentrifugal field. This is of great advantage when concentration isnecessary. Also, the centrifugal force makes the drying process muchmore efficient, and material does not fly off the evaporation surfacedue to bumping or boiling.

The column fittings can be arranged so that columns may be prepacked andattached to the chromatograph rotor, or may be changed as necessary.

Numerous characteristics, advantages and embodiments of the inventionhave been described in detail in the foregoing description withreference to the accompanying drawings. However, the disclosure isillustrative only and the invention is not limited to the preciseillustrated embodiments. Various changes and modifications may beeffected therein by one skilled in the art without departing from thescope or spirit of the invention.

I claim:
 1. A centrifugal fast chromatograph for automaticallyperforming, monitoring, and comparing multiple sample elutions,comprising:a chromatograph rotor; a rotor drive for rotating saidchromatograph rotor; a plurality of chromatographic columnsequiangularly mounted on said chromatographic rotor, saidchromatographic columns each comprising an optical flow cell havingtransparent windows disposed at one end of said chromatographic column;a sample holding transfer disk located at the center of saidchromatographic rotor, said transfer disk having a plurality of samplewells equiangularly disposed around the periphery of said transfer disk,each of said sample wells corresponding to a respective saidchromatographic column; first and second distributor rings coaxiallymounted on said chromatograph rotor; a fluid carrying center lineextending from said transfer disk via said first distributor ring to theinner end of said chromatographic column and having a center line valveto direct fluid to an exit drain; a fluid carrying edge line extendingfrom said outer end of said chromatographic column to said seconddistributor ring and having an edge line valve to direct fluid to anexit drain; a gradient maker in fluid communication with saidchromatographic columns through both said center line and said edge lineto provide a liquid gradient for column elution and a regeneratingsolution for column regeneration; stream apportioning bladesequiangularly disposed on the inner periphery of said chromatographrotor for directing solution from said sample wells and a liquidgradient stream from said gradient maker to said chromatographiccolumns; a valve system for reversing flow through the column whilemaintaining centrifugally-generated pressure and flow; a light sourcemounted on one side of said chromatograph rotor oriented to shine alight beam through said optical flow cells; a photodetector mounted onthe side of said chromatograph rotor opposite said one side, andopposite said light source to detect light shone through said opticalflow cells; and a microprocessor type computer for controlling saidgradient maker, and said chromatograph rotor through said rotor drive,for controlling the chromatographic process, for monitoring thechromatographic process, and for providing real time CRT display of allchromatograph columns.
 2. The centrifugal fast chromatograph as setforth in claim 1 wherein said photodetector comprises a photomultiplier.3. The centrifugal fast chromatograph as set forth in claim 1 whereinsaid center and edge lines are sloped and further comprising a trap,said sloped feed lines and said trap preventing said chromatographiccolumns from running dry.
 4. The centrifugal fast chromatograph as setforth in claim 1 wherein said optical flow cell has a long optical flowpath to increase sensitivity of detection, and in which fluid mixing andloss of resolution are prevented by centrifugal force acting on a liquiddensity gradient.
 5. The centrifugal fast chromatograph as set forth inclaim 1 further comprising distributor rings and wherein said gradientmaker is in fluid communication with said center line and said edge linethrough respective gradient feed lines, and respective distributorrings, each said distributor ring containing apportioning blades.
 6. Thecentrifugal fast chromatograph as set forth in claim 5 furthercomprising a sample measuring and transfer system to load measured andidentified samples into the sample wells of said transfer disk at rest.7. The centrifugal fast chromatograph as set forth in claim 1 whereinsaid center line valves and respective said edge line valves areinterconnected and operate simultaneously.
 8. The centrifugal fastchromatograph as set forth in claim 7 wherein said interconnected centerline and edge line valves are pressure operated valves and comprisevalve pairs, said valve pairs comprising:a dual headed piston havingfirst and second head portions connected by an elongate member, eachsaid head portion having an opening through its center to permit thepassage of fluid, said first opening providing a fluid passageway fromsaid center line to a drain and said second opening providing a fluidpassageway from said edge line to a drain; and biasing means for biasingsaid piston in a rest position preparatory to elution.
 9. Thecentrifugal fast chromatograph as set forth in claim 8 wherein saidbiasing means comprises a spring.
 10. A centrifugal chromatographcomprising:a plurality of chromatographic columns rotatable about anaxis; a fluid carrying center line for each chromatographic column beingin flow communication with an inner end of the column; a fluid carryingedge line for each chromatographic column being in flow communicationwith an outer end of the column; and valve means for the center linesand the edge lines for selectively controlling fluid flow through thecolumn.
 11. The centrifugal chromatograph of claim 10, wherein a liquidgradient to be introduced into the chromatographic column is of varyingdensity allowing gradient density stabilization to prevent fluid mixingor loss of resolution.
 12. The centrifugal chromatograph of claim 11further comprising stream apportioning blades for directing fluids tothe chromatographic columns.
 13. The centrifugal chromatograph of claim12 further comprising distributor rings and wherein a gradient maker isin fluid communication with said center line and said edge line throughthe respective distributor rings, each said distributor ring containingapportioning blades.
 14. The centrifugal chromatograph of claim 13further comprising a sample measuring and transfer system to loadmeasured and identified samples into the sample wells of said transferdisk.
 15. The centrifugal chromatograph of claim 10, further comprisingantiregurgitation means for each fluid carrying center line and eachfluid carrying edge line for preventing regurgitation of fluidsintroduced into said chromatographic columns.
 16. The centrifugalchromatograph of claim 15, wherein said antiregurgitation means includesupwardly sloping portions of each center line and each edge line. 17.The centrifugal chromatograph of claim 15, wherein saidantiregurgitation means comprises a trap positioned between an inletportion of each center line and each edge line and the respectivecolumn.
 18. The centrifugal chromatograph of claim 10, further includingdrain means for each center line and each edge line, said drain meansbeing located inboard of the columns for preventing the column fromrunning dry.
 19. The centrifugal chromatograph as of claim 10, furthercomprising gradient maker means for producing a liquid gradient ofvarying density for allowing gradient stabilized reversible two-waycentrifugation.
 20. The centrifugal chromatograph of claim 19, whereinthe gradient maker is in fluid communication with the chromatographiccolumns selectively through the center lines and the edge lines forintroducing the liquid gradient to elute or regenerate thechromatographic columns.
 21. The centrifugal chromatograph of claim 20,wherein the gradient maker includes reversing means for selectivelyalternating between the center lines and the edge lines for introducingthe liquid gradient into the chromatographic columns.
 22. Thecentrifugal chromatograph of claim 10, wherein the valve means comprisesa tandem valve for each column, said tandem valve coupled in flowcommunication with the center line and edge line.
 23. The centrifugalchromatograph of claim 22, wherein said tandem valve includes a centerline valve and on edge line valve being interconnected to operatesimultaneously.
 24. The centrifugal chromatograph of claim 23, whereinsaid center line valve and said edge line valve open and close therespective line in opposition to the other.
 25. The centrifugalchromatograph of claim 24, wherein said interconnected center line valveand edge line valve are pressure operated valves and comprise valvepairs, said valve pairs comprising:a dual headed piston including firstand second head portions and an interconnecting elongated member, eachsaid head portion having an opening through its center to permit thepassage of fluid, said first opening providing a fluid passageway fromsaid center line to a drain and said second opening providing a fluidpassageway from said edge line to a drain; and biasing means for biasingsaid piston in a rest position preparatory to elution.
 26. Thecentrifugal chromatograph of claim 25, wherein said biasing meanscomprises a spring.
 27. The centrifugal chromatograph of claim 10,further comprising an optical flow cell for each chromatographic columnbeing rotatable therewith, each flow cell having a transparent windowdisposed at one end of said column.
 28. The centrifugal chromatograph ofclaim 27 further comprising a light source oriented to shine a lightthrough each optical flow cell as it passes a point in a circular pathof rotation and a photodetector oriented to detect light shone througheach optical flow cell, wherein the light source and photodetector aremounted on opposite sides of the path.
 29. The centrifugal chromatographof claim 28, wherein each optical flow cell has a relatively long radialoptical flow path to increase the sensitivity of detection of thephotodetector.
 30. The centrifugal chromatograph of claim 28, whereinsaid photodetector comprises a photomultiplier.
 31. The centrifugalchromatograph of claim 10 further comprising a sample holding transferdisk located at the center of the chromatograph having a plurality ofsample wells disposed around the periphery of said transfer disk witheach of the sample wells corresponding to a respective chromatographiccolumn.